Measuring prions causing bovine spongiform encephalopathy or chronic wasting disease by immunoassays and transgenic mice (original) (raw)
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Prion
Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases that are based on the misfolding of a cellular prion protein (PrP(C)) into an infectious, pathological conformation (PrP(Sc)). There is proof-of-principle evidence that a prion vaccine is possible but this is tempered with concerns of the potential dangers associated with induction of immune responses to a widely-expressed self-protein. By targeting epitopes that are specifically exposed upon protein misfolding, our group developed a vaccine that induces PrP(Sc)-specific antibody responses. Here we consider the ability of this polyclonal antibody (SN6b) to bind to a mutant of PrP(C) associated with spontaneous prion disease. Polyclonal antibodies were selected to mimic the vaccination outcome and also explore all possible protein conformations of the recombinant bovine prion protein with mutation T194A [bPrP(T194A)]. This mutant is a homolog of the human T183A mutation of PrP(C) that is associated ...
The American journal of pathology, 1998
Antibodies to the prion protein (PrP) have been critical to the neuropathological and biochemical characterization of PrP-related degenerative diseases in humans and animals. Although PrP is highly conserved evolutionarily, there is some sequence divergence among species; as a consequence, anti-PrP antibodies have a wide spectrum of reactivity (from strong immunopositivity to lack of reactivity) when challenged with PrP from diverse species. We have produced an antibody (anti-PrP95-108) raised against a synthetic peptide corresponding to residues 95 to 108 of human PrP and have characterized it by epitope mapping, Western immunoblot analysis, and immunohistochemistry. The antibody recognizes not only human PrP isoforms but also pathological PrP from all species tested (ie, cattle, sheep, hamsters, and mice). This is probably due to the fact that the epitope recognized by this antibody includes residues 100 to 108 of human PrP, a sequence that is also present in PrP of several other ...
Establishment of a Chicken Monoclonal Antibody Panel Against Mammalian Prion Protein
Journal of Veterinary Medical Science, 2004
A panel of chicken monoclonal antibodies (mAbs) was developed against prion protein (PrP), the sequence of which is a highly conserved molecule among mammals. A portion of the splenocytes from chickens immunized with recombinant mouse PrP was fused with the chicken B cell line, MuH1. The remaining splenocytes were used to generate the recombinant mAbs by phage display. A t otal of 36 anti-PrP mAbs, 2 from cell fusion and 34 from phage display were established. The specificity of these mAbs was determined by Western blot and ELISA using various PrP antigens including recombinant PrPs, synthetic PrP peptides and PrPs from brains or scr apieinfected neuroblastoma cell line. These mAbs were classified into three main groups, protease K (PK)-sensitive (Group I), PK cl eavage site proximal (Group II) and PK-resistant (Group III), based on their abilities to recognize PrP following PK-treatment. Some mAbs were found to selectively recognize different glycoforms of PrP as well as the metabolic fragments of PrP. Furthermore, we found that PrP recognition by chickens differed from that by PrP-knockout mouse. These results indicate that these newly generated PrP ant ibodies from chickens will help to research the PrP and to establish the diagnosis of prion disease.
PrP-specific camel antibodies with the ability to immunodetect intracellular prion protein
Journal of General …, 2010
Although there is currently no effective treatment for prion diseases, significant advances have been made in suppressing its progress, using antibodies that block the conversion of PrP C into PrP Sc. In order to be effective in treating individuals that have prion diseases, antibodies must be capable of arresting disease in its late stages. This requires the development of antibodies with higher affinity for PrP Sc and systems for effective translocation of antibodies across the bloodbrain barrier in order to achieve high concentrations of inhibitor at the site of protein replication. An additional advantage is the ability of these antibodies to access the cytosol of affected cells. To this end, we have generated PrP-specific antibodies (known as PrioV) by immunization of camels with murine scrapie material adsorbed to immunomagnetic beads. The PrioV antibodies display a range of specificities with some recognizing the PrP 27-30 proteinase K-resistant fragment, others specific for PrP C and a number with dual binding specificity. Independent of their PrP conformation specificity, one of the PrioV antibodies (PrioV3) was shown to bind PrP C in the cytosol of neuroblastoma cells. In marked contrast, conventional anti-PrP antibodies produced in mouse against similar target antigen were unable to cross the neuronal plasma membrane and instead formed a ring around the cells. The PrioV anti-PrP antibodies could prove to be a valuable tool for the neutralization/clearance of PrP Sc in intracellular compartments of affected neurons and could potentially have wider applicability for the treatment of so-called protein-misfolding diseases.
Journal of General Virology, 2009
To evaluate further the reactivity of prion-specific monoclonal antibodies containing the 89-112 or 136-158 prion protein (PrP) polypeptides, immunoprecipitations were performed on brain extracts from Italian bovines, sheep and goats with transmissible spongiform encephalopathies. No binding of IgG 89-112 or IgG 136-158 to PrP in normal brain extracts was detected. Conversely, both reagents immunoprecipitated PrP from bovine and bovine amyloidotic spongiform encephalopathies, and from typical and atypical scrapie brain extracts. The immunoprecipitated PrP bands mirrored the Western blot (WB) profile of the different prion strains, indicating universal affinity of two independent PrP regions for disease-associated PrP conformers regardless of species source and strain properties. Immunoprecipitation with motif-grafted antibodies increased the sensitivity of conventional detection methods based on centrifugation followed by WB, which was confirmed by assay of diluted samples using both methods. These reagents or derivative molecules may thus find broad applications in prion detection and research.
Generation of antibodies against prion protein in wild-type mice via helix 1 peptide immunization
Journal of Neuroimmunology, 2003
We present here the development of antibodies against prion protein in BALB/C mice using as antigen human helix 1 of PrP. This sequence is suggested to be involved in protein pathological conformational changes, and is distinguished from that of mice by one amino acid. The immune tolerance to an 'almost-self' epitope and the poor immunogenicity of short peptides was overcome by using Multiple Antigen Peptide displaying eight copies of helix 1. The generated antibodies recognize the whole prion protein with a high binding constant and the established protocol may lead to an active immunization towards therapeutics of prion disease. D
Conformation-Dependent High-Affinity Monoclonal Antibodies to Prion Proteins
The Journal of Immunology, 2010
Prion diseases are fatal, neurodegenerative illnesses caused by the accumulation of PrP Sc , an aberrantly folded isoform of the normal, cellular prion protein. Detection of PrP Sc commonly relies on immunochemical methods, a strategy hampered by the lack of Abs specific for this disease-causing isoform. In this article, we report the generation of eight mAbs against prion protein (PrP) following immunization of Prnp-null mice with rPrP. The eight mAbs exhibited distinct differential binding to cellular prion protein and PrP Sc from different species as well as PrP-derived synthetic peptides. Five of the eight mAbs exhibited binding to discontinuous PrP epitopes, all of which were disrupted by the addition of 2-ME or DTT, which reduced the single disulfide bond found in PrP. One mAb F20-29 reacted only with human PrP, whereas the F4-31 mAb bound bovine PrP; the K D values for mAbs F4-31 and F20-29 were ∼500 pM. Binding of all five conformation-dependent mAbs to PrP was inhibited by 2-ME in ELISA, Western blots, and histoblots. One conformation-dependent mAb F4-31 increased the sensitivity of an ELISA-based test by nearly 500-fold when it was used as the capture Ab. These new conformation-dependent mAbs were found to be particularly useful in histoblotting studies, in which the low backgrounds after treatment with 2-ME created unusually high signal-to-noise ratios.
Journal of Veterinary Diagnostic Investigation, 2006
Confirmatory diagnosis of prion diseases in humans and animals relies on the histopathological examination and immunodetection of the protease-resistant isoform of prion protein (PrP res). The generation of novel PrP-specific monoclonal antibodies (MAbs) has greatly improved diagnostic methodology and basic research on prion diseases as well. In this study, the performance of 3 different PrP-specific MAbs in recognizing brain PrP res deposits from cows affected with bovine spongiform encephalopathy (BSE) was compared by using a standard immunohistochemical technique under different pretreatment conditions. All antibodies showed similar reactivity after denaturing treatment. However, greater differences were found among them after proteinase K treatment, even in the absence of a denaturing step. In fact, 1 MAb (2A11) was able to react with PrP res deposits in the absence of a denaturing step, yielding the strongest signal and confirming the usefulness of MAb 2A11 in immunohistochemistry for the diagnosis of BSE.